The exploitation of cellular disease targets is particularly challenging for the most ubiquitous target class, protein-protein interactions, where large and shallow interaction surfaces often render inhibition by small molecules a major challenge (Surade & Blundell (2012), Chem. Biol. 19: 42-50). While peptides have proven an effective alternative for some protein-protein extracellular receptors, their role has been limited as they typically cannot enter cells and are inherently unstable in vivo. However, nature uses disulfide bond constrained peptide structures in the form of peptide toxins and knottins to improve proteolytic and thermal stability (Terlau et al. (2004), Physiol. Rev. 84: 41-68; Daly et al. (2011), Curr. Opin. Chem. Biol. 15: 362-368; Clark et al. (2010), Angewandte Chemie, 49: 6545-6548). These highly constrained peptides leverage the fact that proteases can only recognize and break down unfolded peptides. This concept was extended with the development of peptides “stapled” into an alpha-helical shape using optimized cross-linking chemistry, mimicking the structure found at the interface of many protein-protein interactions (Walensky et al. (2004), Science 305: 1466-1470). The resulting molecules possess improved biological properties such as cell penetration (Verdine et al. (2012), Methods in Enzymology, Protein Engineering for Therapeutics, Academic Press: 3-30). However, limitations have hindered their use as therapeutics. These include moderate cell potencies in the high nM to low uM range resulting in high doses required for efficacy, which combined with the hydrophobic nature of their interaction surfaces, produces sub-optimal physicochemical properties such as solubility.
Irreversible inhibitors that covalently bind to their target protein have been described in the art (Singh et al. (2011), Nature Rev. Drug Discovery 10: 307-317; Barf and Kaptein, (2012), J. Med. Chem. 55: 6243-6262). Covalent irreversible inhibitors of drug targets have a number of important advantages over their reversible counterparts as therapeutics. Prolonged suppression of the drug targets may be necessary for maximum pharmacodynamic effect and an irreversible inhibitor can provide this advantage by permanently eliminating existing drug target activity, which will return only when new target protein is synthesized. When an irreversible inhibitor is administered the therapeutic plasma concentration of the irreversible inhibitor would need to be attained only long enough to briefly expose the target protein to the inhibitor, which would irreversibly suppress activity of the target and plasma levels could then rapidly decline while the target protein would remain inactivated. This irreversible binding has the potential advantage of lowering the minimal blood plasma concentration at which therapeutic activity occurs, minimizing multiple dosing requirements and eliminating the requirement for long plasma half-lives without compromising efficacy. All of these considerations could reduce toxicity due to any nonspecific off-target interactions that may occur at high or prolonged blood plasma levels. Irreversible inhibitors also likely have advantages in overcoming drug resistance requirements in two ways. First irreversible inhibitors eliminate the requirement for long blood plasma half-lives without compromising efficacy. Second, while resistance mutations can compromise non-covalent binding and reduce non-covalent affinity, it is still possible to inactivate the target through irreversible inhibition. Peptidomimetic macrocyclic irreversible inhibitors have several important advantages relative to stapled peptides. First, not having the necessity for long plasma half-lives is particularly advantageous since in the design of stapled peptides optimizing for proteolytic stability to prolong half-lives is crucial to ensure sufficient plasma coverage of the target protein to elicit a sustained therapeutic response. Second, irreversible inhibitors enhance the potency (measured as the IC50 over a fixed time period), which may result in a lower dose of inhibitor required to silence the target protein hence mitigating formulation issues and not exacerbating physicochemical properties such as solubility.
Many reversible inhibitors of proteins are presently known, as are the binding sites in the proteins to which the reversible inhibitors bind. The binding sites of these reversible inhibitors are sometimes populated with amino acids that are capable of covalent modification with suitably reactive ligands. In other instances, amino acids are located near the binding sites of reversible inhibitors that are capable of covalent modification with suitably reactive ligands. Amino acids capable of covalent modification are typically those, which have a heteroatom such as O, S, or N in the side chain such as threonine, cysteine, histidine, serine, tyrosine and lysine. Sulfur is amenable to covalent modification due to the nucleophilicity of sulfur and as such there are numerous examples of ligands that modify cysteine in proteins of interest. Amino acids such as lysine are usually sufficiently unreactive that ligands do not react in vivo with lysine. However, it is known that a hydrogen bond donor amino acid proximal to lysine can enhance the nucleophilicity of the lysine nitrogen by lowering the pKa making it more amenable to react with electrophilic warheads (US Patent Application No: US 2011/0269244 A1). Amino acids with hydrogen donor capability are arginine, threonine, serine, histidine, tyrosine and lysine. In some cases the hydrogen bond donation, either by a side chain or even a main chain amide can, in many cases, enhance the electrophilicity of a warhead. When such a hydrogen bond donor is also positively charged, Coulombic attraction can accelerate the reaction, for example, by stabilizing the formation of an enolate as in the example of an acrylamide. The present invention addresses these limitations in the art by the design of peptidomimetic macrocycles incorporating an amino acid warhead designed to be proximal to a lysine or cysteine amino acid of the target protein to form a covalent bond resulting in irreversible inhibition of the target protein.
BCL2-A1 (BFL-1) (Vogler et al. (2012), Cell Death Diff. 19: 67-74) and MCL-1 are proteins in the B-cell lymphoma 2 (BCL2) target family (Bajwa et al. (2012), Expert Opin. Ther. Patents 22: 37-55) whose anti-apoptotic members have been identified as important cellular oncogenes that not only promote tumorigenesis but also contribute to chemotherapeutic drug resistance. The potential of this target class is highlighted by ABT-263, a BCL2 family inhibitor helix mimetic in multiple combination clinical trials with existing oncology drugs (Tse et al. (2008), Cancer Research 68: 3421-3428). This class of compound shows significant potential but suffers from toxicity (thrombocytopenia) due to inhibition of off-target BCL2 pathways (BCL-XL) (Bajwa et al. (2012), Expert Opin. Ther. Patents 22: 37-55) and the emergence of resistance. There is evidence that overexpression of BCL2-A1 and upregulation of MCL-1 are the primary resistance mechanisms for Abbott's BCL2 clinical inhibitors (Vogler et al. (2009), Blood, 113: 4403-4413; Al-Harbi et al. (2011), Blood, 118: 3579-3590), with studies suggesting that it may be possible to screen for these using biomarkers (Al-Harbi et al. (2011), Blood, 118: 3579-3590). In addition BCL2-A1 protein is overexpressed in a variety of cancer cells, and increased BCL2-A1 expression in advanced tumor stages has been noted in a number of studies (Vogler et al. (2012), Cell Death Diff. 19: 67-74; Piva et al. (2006), J. Clin. Invest. 116: 3171-3182). It has also been shown that BCL2-A1 down regulation sensitizes non-small cell lung cancer (NSCLC) to gemcitabine (Kim et al. (2011), Molecular Cancer, 10: 1-16) while also exhibiting NPM-ALK induced upregulation in anaplastic large cell lymphomas (ALCLs) which can be extrapolated to ALK modulated NSCLC data in the clinic. BCL2A1 was recently identified as a lineage-specific antiapoptopic oncogene that confers resistance to BRAF inhibition (Haq et al. (2013), PNAS, 110: 4321-4326).